The global HIV/AIDS pandemic is in its 30th year, yet infection rates remain alarmingly high, with 2.6 million people worldwide having become infected in 2009. These troubling statistics highlight the need for efficacious antiretroviral chemoprophylaxis as a preventative measure for at-risk populations. Two large-scale clinical trials recently have shown that pre-exposure prophylaxis (PrEP) prevents infection in a significant proportion of individuals. The CAPRISA 004 trial showed that HIV-susceptible women who adhered to a regimen involving the application of 1% tenofovir vaginal gel prior to intercourse achieved a 54% reduction in HIV transmission. In the iPrEx trial, men and transgender women who have sex with men receiving once-daily emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) achieved a 44% reduction in HIV acquisition. These highly encouraging results provide a clear rationale for future clinical studies based on systemic and topical PrEP, likely using a combination of antiretroviral agents such as TDF and FTC. In preparation for these trials, the NIAID has identified the development of novel ex vivo systems for the evaluation of antiretroviral agents as a key priority area. Antiretroviral drugs have unpredictable pharmacokinetic (PK) properties involving extensive drug metabolism and transport by membrane-associated carrier proteins. Combination drug therapy often introduces complex drug-drug interactions that can result in toxic or sub-therapeutic drug concentrations and compromise treatment. The objective of this application is to establish cell culture models that faithfully recreate the membrane drug transport processes in the vaginal epithelium and to use these systems to study the transcellular transport of the TDF-FTC drug combination ex vivo. The permeability of biological membranes is one of the most important determinants of a drug's PK profile. Drugs pass through cells by a combination of passive transcellular and carrier-mediated processes. Given the paucity of knowledge on the expression of molecular transporters in the genital tract of humans and animal models, representative ex vivo studies predictive of in vivo PK currently are not possible. We propose to address this urgent need through the following three research phases: (1) Measure baseline gene expression of molecular transporters (membrane proteins) in vaginal tissue from humans and animal models used in the evaluation of topical microbicide formulations; (2) Develop an ex vivo model and apply it to the TDF-FTC combination through the creation of novel, and the utilization of existing, vaginal cell culture systems; and (3) Evaluate ex vivo model predictions by conducting in vivo PK studies using intravaginal rings delivering TDF and FTC topically to the genital tract. PUBLIC HEALTH RELEVANCE: The HIV/AIDS pandemic is in its 30th year, yet infection rates in the global population remain alarmingly high, with 2.6 million people having become infected in 2009. The prophylactic use of antiviral drugs has been shown to reduce HIV infection rates in susceptible individuals, suggesting that future large-scale clinical studies will be based on prophylaxis using combinations of multiple drugs, but these are known to become prematurely metabolized and interact with each other to compromise their effectiveness. The proposed research will develop cell culture models simulating the mammalian vaginal epithelium to test antiviral drugs administered topically, thus paving the way for clinical trials of these therapeuti agents with the goal of empowering women to protect themselves from HIV acquisition.